Cardiovascular disease (CVD) continues to be a leading cause of mortality and morbidity worldwide. We have previously demonstrated that mitochondrial DNA copy number (mtDNA-CN), which reflects the high degree of variation in the number of mitochondrial genomes per cell (10s to 100s), is a novel risk factor for CVD. In a prospective cohort analysis including 21,870 participants, a 1 standard deviation decrease in mtDNA-CN was associated with a 1.23 (95%CI 1.19 to 1.26) increased risk of CVD. Moreover, a direct clinical utility was demonstrated, with the addition of mtDNA-CN to the 2013 ACC/AHA Pooled Cohorts Equations for estimating 10-yr atherosclerotic CVD risk significantly improving both sensitivity and specificity for the recommendations on initiating statin therapy. While mtDNA-CN captures the quantity of mitochondria, and higher mtDNA-CN levels are associated with increased mitochondrial function this measurement does not take into account the quality of the mitochondria. Mitochondrial DNA heteroplasmy, which is universally observed and accumulates with age, is likely to have negative consequences for mitochondrial function. Thus, we hypothesize that the effect of mtDNA- CN on CVD risk will be directly impacted by levels of heteroplasmy, and that higher levels of heteroplasmy will be an independent risk factor for CVD. To test this hypothesis, we will first use the combined ARIC, CHS, FHS, MESA and RS cohorts (n=23,954; 6,048 incident CVD events), to determine the association of baseline measures of mtDNA heteroplasmy with incident CVD. Second, changes in mtDNA heteroplasmy may reflect ongoing disease processes (e.g. inflammation), and thus we will use DNA collected from the initial baseline visit along with up to 2 follow-up visits in each cohort (n=7,364, 3,430 incident CVD events) to assess how longitudinal changes in mtDNA heteroplasmy influence future CVD risk. Third, one potential consequence of decreased mitochondrial function arising from somatic mutation is an upregulation in the production of mitochondria. Therefore, high mtDNA-CN in the presence of high heteroplasmy may actually be harmful rather than protective. Thus, we propose to combine these measures and assess their role in improving the ACC/AHA risk score. Finally, we will functionally characterize the consequences of mtDNA heteroplasmy by introducing and manipulating the levels of specific mtDNA genetic variants through mitoTALENs. We will further explore a plausible biological mechanism testing the impact of heteroplasmy on nuclear DNA methylation and gene expression, both of which are modified by mtDNA-CN. This proposal leverages 5 well-established cohorts, with ~6000 cases of incident CVD in over 20 years of follow-up, together with a comprehensive set of functional validation experiments, to elucidate the role of mtDNA heteroplasmy in CVD risk. Both heteroplasmy and mtDNA- CN are readily measured in whole blood, a highly relevant tissue that plays a primary role in the development of CVD. More importantly, these two measures represent a novel class of risk factors not captured by any of the established CVD risk factors and therefore opens the potential for novel preventive or therapeutic strategies.